Chronic viral infections such as HIV, HBV, and HSV afflict enormous suffering, life loss, and financial burdening among the infected individuals. These infectious diseases are incurable, and contagious to variable degrees, and are prominent threats for public health, highlighting the urgent needs for curative therapies. To date, effective antiviral therapies only suppress viral replication but do not clear virus in patients, and do not target the viral genetic materials of latently integrated (proviral DNA) or non-replicating episomal viral genomes (such as cccDNA) in human cells. Nevertheless, these viral DNAs have been reported to directly cause these chronic or latent infections.
Recent breakthroughs in biotechnology have identified several molecular gene editing tools such as zinc finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), homing endonucleases (HEs), and most notably the clusters of regularly interspaced short palindromic repeat (CRISPR)-associated protein Cas9. These nucleases are highly specific, DNA-cleaving enzymes, and have been recently demonstrated as potential therapeutic applications to eradicate HBV, HIV, HSV, and herpesvirus by targeting the enzymatic systems directly to essential viral genome sequences.
Both ZFNs and TALENs are composed of protein-based programmable, sequence-specific DNA-binding modules and nonspecific DNA cleavage domains, which make multiple-site targeting extremely challenging. This is even more challenging for HEs, which have the same protein domains for both DNA binding and cleavage.
Unlike nucleases such as ZFNs, TALENs, and HEs, CRISPR-Cas-RNA complexes contain short CRISPR RNAs (crRNAs), which direct the Cas proteins to the target nucleic acids via Watson-Crick base pairing to facilitate nucleic acid destruction. Three types (I-III) of CRISPR-Cas systems have been functionally identified across a wide range of microbial species. While the type I and III CRISPR systems utilize ensembles of Cas proteins complexed with crRNAs to mediate the recognition and subsequent degradation of target nucleic acids, the type II CRISPR system recognizes and cleaves the target DNA via the RNA-guided endonuclease Cas9 along with two noncoding RNAs, the crRNA and the trans-activating crRNA (tracrRNA). The crRNA::tracrRNA complex directs Cas9 DNA endonuclease to the protospacer on the target DNA next to the protospacer adjacent motif (PAM) for site-specific cleavage (FIG. 1). This system is further simplified by fusing the crRNA and tracrRNA into a single chimeric guide RNA (sgRNA) with enhanced efficiency, and can be easily reprogrammed to cleave virtually any DNA sequence by redesigning the crRNAs or sgRNAs.
The CRISPR/Cas9 system has been evaluated as potential therapeutic strategy to cure chronic and/or latent viral infections such as HIV, HBV, and Epstein-Barr virus (EBV). Hu and et al. reported CRISPR/Cas9 system could eliminate the integrated HIV-1 genome by targeting the HIV-1 LTR U3 region in single and multiplex configurations. It inactivated viral gene expression and replication in latently infected microglial, promonocytic, and T cells, completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs, and caused neither genotoxicity nor off-target editing to the host cells. Yang and et al. reported the CRISPR/Cas9 system could significantly reduce the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector, and disrupt the HBV expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection. They observed that two combinatorial gRNAs targeting different sites could increase the efficiency in causing indels. The study by Seeger and Sohn also reported CRISPR/Cas9 efficiently inactivated HBV genes in NTCP expressing HepG2 cells permissive for HBV infection. Wang and Quake observed patient-derived cells from a Burkitt's lymphoma with latent Epstein-Barr virus infection presented dramatic proliferation arrest and a concomitant decrease in viral load after exposure to a CRISPR/Cas9 vector targeted to the viral genome and a mixture of seven guide RNAs at the same molar ratio via plasmid.
In spite of initial success in evaluation of CRISPR/Cas9 for therapeutic applications in treatment of chronic or latent viral infections, off-target cleavage is a major concern for any nuclease therapy. Studies indicated that a 10-12 bp “seed” region located at the 3′-end of the protospacer sequence is critical for its site-specific cleavage, and that Cas9 can tolerate up to seven mismatches at the 5′-end of the 20-base protospacer sequence.
Cas9::sgRNA was reported to be a single turnover enzyme, and was tightly bound by the cleaved DNA products. In thermodynamic term, the energetic increase in the unwound DNA helix (two DNA single strand) in Cas9 is compensated by free energy decrease of the hybridization between DNA and crRNA and of the binding of Cas9 with the formed DNA:crRNA helix and with the resulting single strand DNA; to release the cleaved DNA products, especially the hybridized DNA strand with crRNA, the free energy increase in the released DNA requires compensation of free energy decrease by certain processes such as binding of additional protein factor(s), enabling the recycling of the Cas:: sgRNA enzyme, which is believed to happen under physiological conditions. A more related but also more complicated in vitro assay comprising both Cas9::sgRNA and other binding molecules or cellular assay is needed for reliable SAR based on multiple turnover enzymatic reactions.
Another major concern is the requirement of more sophisticated approaches to delivery. To date, the Cas9 protein and RNAs (either dualRNA (crRNA/tracrRNA) or a single guide RNA (sgRNA, ˜100 nt)) have been mostly introduced by plasmid transfection either as whole or separately, which makes chemical modifications of RNAs extremely challenging, if not impossible, and also is limited by random integration of all or part of the plasmid DNA into the host genome and by persistent and elevated expression of Cas9 in target cells that could lead to off-target effects. A recent study presented lipid-mediated delivery of unmodified Cas9::sgRNA complexes using common cationic lipid nucleic acid transfection reagents such as Lipofectamine resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection, and be effective both in vitro and in vivo. Another example presented treatment with cell penetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs led to endogenous gene disruptions in human cell lines with lower off-target mutation frequencies than plasmid transfection.
To minimize or completely eradicate off-target effects, and to overcome other major obstacles to its pharmaceutical applications including the lack of stability of RNA, low potency of Cas9-gRNA at the target sites, large sizes of Cas9 proteins (˜150 kDa), chemically modified sgRNAs may provide a very effective strategy as indicated by the therapeutic application of antisense oligonucleotides and progress in small interfering RNA technology, and also supported by a recent report on chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA) in combination with unmodified transactivating crRNA (tracrRNA), showing a comparable efficacy as sgRNA, though as dual guide RNAs, this combination should be less effective than sgRNA incorporated with same chemical modifications because of entropic and other factors. However, considering the size of sgRNA (˜100 nt), large scale chemically manufacturing such large molecules is industrially challenging and costly. Methods for preparation of long RNAs (sgRNA) compatible to extensive chemical modifications and/or significantly truncated sgRNAs or crRNA/tracrRNAs of lengths compatible to current industrial RNA chemical synthesis are in need. Truncating sgRNAs or crRNA/tracrRNAs is limited because of the many essential binding interactions between Cas9 and sgRNA and the complicated molecular mechanisms of recognition and cleavage of target DNAs. A possible solution can be based on recent progress in chemical ligations of nucleic acids. Brown and et al. showed the CuAAC reaction (click chemistry) in conjunction with solid-phase synthesis could produce catalytically active hairpin ribozymes around 100 nucleotides in length.
Recent disclosure of the structure of CRISPR/Cas9-sgRNA complexes indicated that the linkloop (tetraloop) of the sgRNA protrudes outside of the CRISPR/Cas9sgRNA complex, with the distal 4 base pairs (bp) completely free of interactions with Cas9 peptide side chains (FIG. 2). The natural guide RNAs comprise CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), directing Cas9 to a specific genomic locus harboring the target sequence, in spite of their lower efficacy than sgRNA. Therefore this tetraloop can be replaced by small molecule non-nucleotide linkers (nNt-linker), and sgRNA can be divided into two pieces of 30-32 nt (crgRNA) and ˜60 nt (tracrgRNA) (FIG. 3), respectively, or into three pieces (˜30-32 nt (crgRNA), ˜30 nt (tracrgRNA1), and ˜30 nt (tracrgRNA2)), or multiple pieces based on the void of interactions between certain other sections of sgRNA and Cas9, and joined by chemical ligations in vitro as ligated guide RNAs (lgRNA). These RNAs (crgRNA and tracrgRNA) are more readily synthesized chemically at industrial scale, and can even be further shortened by introductions of chemical modifications at various sites, and therefore more commercially accessible, and lgRNAs can be optimized by chemical modifications for better efficacy and specificity and for targeted delivery.
As presented by previous studies, targeting viral DNA at multiple sites could enhance the effectiveness, which can be better practiced by delivering whole CRISPR/Cas9-lgRNA complexes composed of chemical libraries of different crgRNAs (including various spacers) and formed in vitro. This chemical ligation strategy can provide diverse chemically modified lgRNAs targeting multiple sites and/or variants/mutations of a single site in viral genomes equivalent to combination therapies such as HAART.